The culturing of mammalian cells presents many challenges as compared to bacteria, yeast and molds. Bacterial-type cells have tougher outer cell walls and can be manipulated without injuring the cells during culture, whereas mammalian cells are delicate and cannot withstand stress. Bacterial-type cells are not organized into functional tissue groups with cell differentiation or organization as seen in higher life forms such as mammals.
Mammalian tissue can be grouped into three general categories of (1) organ tissue (2) structural tissue and (3) blood producing tissue. Mammalian tissue is composed of aggregates of cells that share a functional interrelationship in order to have tissue growth. Mammalian tissue is composed of different types of cells characterized by different morphology and immunochemical properties. Cellular differentiation in mammals involves complex interactions in which cell membrane junctions, extracellular matrices such as basement membrane and ground substances. Soluble signals produced and shared among the cells play an important role.
Study of normal mammalian tissue, however, has been limited because of lack of adequate in vitro culture systems that product tissue of sufficient size and functionality. In contrast, tumor cells are by their nature easier to grow in vitro, possess abnormal growth characteristics and do not behave like normal cells in their interaction with neighboring cells. Sutherland, "Cell and Environment Interactions in Tumor Microregions: The Multicell Spheroid Model", Science, Vol. 240, pp. 177-240, 8 Apr. 1988. Different culture methods for normal and abnormal mammalian cells have been used. Elaborate culture systems for normal mammalian cells have been developed in an attempt to grow normal tissue.
A variety of different cells and tissues, such as bone marrow, skin, liver, pancreas, mucosal epithelium, adenocarcinoma and melanoma, have been grown in culture systems to provide three dimensional growth in the presence of a preestablished stromal support matrix. U.S. Pat. No. 4,963,489, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Oct. 16, 1990; U.S. Pat. No. 5,032,508, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Jul. 16, 1991. A biocompatible, non-living material formed into a three dimensional structure is inoculated with stromal cells. In some cases, the three dimensional structure is a mesh pre-coated with collagen. Stromal cells and the associated connective tissue proteins naturally secreted by the stromal cells attach to and envelop the three dimensional structure. The interstitial spaces of the structure become bridged by the stromal cells, which are grown to at least subconfluence prior to inoculating the three dimensional stromal matrix with tissue-specific cells.
Normal mammalian tissue has been grown for limited periods of time as two-dimensional monolayers on gelled substrate or other surface for anchoring the cells. Adult colonic epithelial cells have been found to be more difficult to culture than fetal colonic epithelial cells. Buset et al. "Defining Conditions to Promote the Attachment of Adult Human Colonic Epithelial Cells", In Vitro Cell. & Dev. Biol., Vol. 23. No. 6 pp. 403-412 (June 1987). To mimic a more normal environment using monolayer culture, cocultures were prepared using two cell types, often with a "feeder layer" of fibroblasts or other cells to supply the primary cells with nutrients and other factors difficult to incorporate into a substrate and to provide the cellular interaction believed to be necessary for the production of differentiated tissue. Reid et al., "Culturing Hepatocytes and Other Differentiated Cells", Hepatology, Vol. 4, No. 3, pp. 548-559 (1984); Haake et al. "Retention of Differentiated Characteristics in Human Fetal Keratinocytes In Vitro", In Vitro Cell. & Dev. Biol., Vol. 25 No. 25 pp. 592-600 (July 1989). Also, monolayers "conditioned" with fibroblast cells have been used to impart into the substrate the soluble growth factors for epithelial cells. Kabalin et al. "Clonal Growth of Human Prostatic Epithelial Cells Is Stimulated by Fibroblasts", The Prostate, Vol. 14, pp. 251-263 (1989). Monolayers do not produce a three dimensional tissue, but rather a two-dimensional spread of cells. Often the cells developed by monolayer culture and coculture become undifferentiated and lack normal function.
Three dimensional in vitro models of differentiated tissue have been produced, however, the cells often do not demonstrate normal cellular activity. Embryonic avian skeletal muscle cells have been grown in vitro on expandable membranes which are gradually and substantially, continuously stretched to simulate the mechanical stimulation of cells experienced in vivo. U.S. Pat. No. 4,940,853, Method for Growing Tissue Specimens in Vitro, Vadenburgh, Jul. 10, 1990. The expandable support membrane supports development of three dimensional structures which more closely resemble tissue grown in vivo, however, normal independent cellular activity was not identified. Additionally, three dimensional human mammary epithelial cells have been grown in collagen. U.S. Pat. No. 5,026,637, Soule, et al., Jun. 25, 1991. However, the cells, under the culture conditions, did not undergo terminal differentiation and cell senescence, but rather were "immortal" in that they retained the capacity to divide forever. Thus, normal cellular activity and naturalization was not observed.
The study of the organ systems exemplifies the complex interrelationship of neighboring cells in mammalian tissue necessary for normal three dimensional tissue growth. Mammalian organ systems have been studied to determine the relationship of the cell types that make up the organ as well as their effects on each other. Studies on normal tissue differentiation have indicated an important interaction between epithelial cells and mesenchymal cells and the production and differentiation of the cells from fibroblasts. Kaye et al., "The Colonic Pericryptal Fibroblast Sheath: Replication, Migration, and Cytodifferentiation of a Mesenchymal Cell System in Adult Tissue" Gastroenterology, Vol. 60. No. 4, pp. 515-536 (1971). Furthermore, small intestine tissue is exemplary of organ tissue types with epithelial cells with functional brush borders and organized columnar cells associated with mesenchymal cells. Other organs have similar organization. Small intestine tissue has physical support in vivo so that the tissue forms crypts which increase the surface area of the organ. In addition to the crypt formation in vivo, there are also immunocytochemical markers for particular cell types with normal functionality as well as cell types that are in developmental phases to produce functional organ tissue.
Immunochemical properties further characterize normal cells types and tissue. Keratin, vimentin and Factor VIII antibodies detect epithelial, fibroblastic and endothelial cells respectively. Villin is a cytoskeletal protein only found in epithelial cells from the small intestine and colon. The angioblast marker is present in certain precursor endothelial cells, particularly dividing cells. Sucrase is an enzyme found in the epithelial cell brush border of the small intestine. Basement membrane and extracellular matrix to components such as laminin, fibronectin, Collagen IV, and proteoglycan are associated with ordering of tissue and cellular polarity. Basement membrane and extracellular matrix development has been related to epithelial-mesenchymal development and differentiation. All these components are necessary for normal tissue growth.
Culture of normal or neoplastic small intestine epithelium as large-scale three dimensional configurations has not been shown with routinely used in vitro culture technology. Chantret et al., "Epithelial Polarity, Villin Expression and Eterocytic Differentiation of Cultured Human Colon Carcinoma Cells: A survey of Twenty Cell Lines" Cancer Res., Vol. 48, pp. 1936-1942 (Apr. 1, 1988). Factors that control proliferation and differentiation in the gastrointestinal tract remain largely unknown. Corps et al., "Stimulation of Intestinal Epithelial Cell Proliferation in Culture by Growth Factors in Human and Ruminant Mammary Secretions" J. Endocr., Vol. 113, pp. 285-290 (3 Nov. 1986); O'Loughlin et al., "Effect of Epidermal Growth Factor on Ontogeny of the Gastrointestinal Tract" Am. J. Physiol., Vol. 249, pp. G674-G678 (8 Jul. 1985); Blay et al., "Contradistinctive Growth Responses of Cultured Rat Intestinal Epithelial Cells to Epidermal Growth Factor Depending on Cell Population Density" J. of Cell. Physiol., Vol. 129, pp.343-346 (15 Jul. 1986).
Cartilage tissue is exemplary of normal structural tissue. Attempts have been made to encourage cartilage formation in vitro by culturing chondrocytes. Typical culture durations achieved in the more successful instances range from 10 days to 30 days, the 30-day point being a transition towards static culture or cellular degeneration and death. The success achieved to date has had limited 3-dimensionality. Typical cell culture methodologies include isolating chondrocytes from various articular surfaces and plating the isolated chondrocytes as a monolayer in a T-flask. Various manipulations were then utilized to encourage chondrocyte expansion and differentiation, and the deposition of proteoglycan into the interstitial matrices. These maneuvers include various growth factor augmentations, an effort to emulate in situ conditions. The extent to which the cells can be maintained in situ is on the order of tens of years as seen in the normal adult. Conversely, in standard cell culture the extent which cells can be maintained in a growth phase or a normal static maintenance phase is on the order of 22 days. It has been demonstrated that multiple cell-surface receptors exist that are important in cellular growth and differentiation, and extracellular matrix formation. Cell surface receptors are labile to destructive cell surface sheer.
Cartilage cells were reportedly cultured for 13 months using a solid substrate in conventional culture vessels. U.S. Pat. No. 4,757,017 Cheung et al., Jul. 12, 1988. The solid substrate was a porous calcium compound. Multilayer cell growth was observed and the cells maintained their phenotype.
Bone marrow has been grown using conventional monolayer cell culture technique. Human bone marrow cell culture technique. Human bone marrow cell production declines over time in monolayer culture. Naughton et. al. disclosed a bone marrow culture system in co-culture with stromal cells or mixtures of cell types on a three dimensional support. U.S. Pat. No. 4,963,489. The three dimensional support was coated by cells such as fetal fibroblasts over a collagen layer on a mesh support.
Mammalian tissue has been grown in bioreactors providing low shear from abnormal cell types such as tumor cells or cocultures with normal and tumor cells. Goodwin et al., "Morphological Differentiation of Colon Carcinoma Cell Lines HT-29 and HT-29K in Rotating-Wall Vessels," In Vitro Cell. Dev. Biol. Vol. 28A, pp. 47-60, January 1992. The bioreactor designs are disclosed in U.S. Pat. Nos. 4,988,623; 5,026,650 and 5,153,131. Co-culture of tumor and normal cells in solid-state culture has been reported as shown in U.S. Pat. No. 4,352,887 to Reid et al. issued Oct. 5, 1982. However, since tumor or abnormal cells grow under conditions that normal cells cannot tolerate, the growth of non-normal cells does not predict success for normal mammalian culture under the same or similar culture conditions.
There are many potential uses of artificially produced tissue for different mammalian systems. For example, small intestine tissue can be used as a model for the molecular and clinical treatment of diseases such as inflammatory bowel disease (Crohn's, ulcerative colitus), malabsorptive syndromes (short-gut syndrome), numerous infectious diseases and tumors of the small bowel. The tissue can be a model for the therapeutic trials prior to in vivo experimentation. However cultures longer than 7 weeks have been difficult to achieve, since crypt cells are unable to survive standard culture regimens, and two-dimensional organ cultures do not support the de novo assembly of stroma and its contribution to epithelial cell growth. Shamsuddin, "Colon Organ Culture as a Model for Carcinogenesis", Colon Cancer Cells, Moyer and Poste, Eds. Academic Press, Inc. 1990.
Also, a mammalian structural tissue such as a cartilage model of high fidelity is important in clinical studies. There are numerous maladies associated with cartilage, including but not limited to knee-joint injuries, back injuries, articular-surface injuries, inflammatory diseases such as arthritis and temporal-mandibular joint disease. Beyond the diseases are the natural processes of maturation through puberty and the geriatric inability to repair and maintain articular surfaces. A tissue model would be beneficial for the analyses and development of therapeutic protocols. Moreover, these models become essential in tailoring pharmaceuticals, i.e.; non-steroidal anti-inflammatories where the pharmaceutical may actually become detrimental to the chondrocyte by way of degradation into secondary metabolites.
The artificial production of functional blood tissue such as normal bone marrow could be used in patients in need of bone marrow replacement which have lost the ability to produce the tissue. The tissue could be grown from a sample and transplanted back to the patient. Also, bone marrow material can be transplanted into a non-autologous recipient.